Method of manufacturing a pharmaceutical composition

ABSTRACT

The present invention relates to crystalline forms of carbetocin, a method of their manufacture, and pharmaceutical compositions thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.16/637,499, filed on Feb. 7, 2020 (now U.S. Pat. No. 10,981,955), whichis the U.S. National Stage of International Application No.PCT/EP2018/071832 filed Aug. 10, 2018, and claims priority to EuropeanPatent Application No. 17186048.9 filed Aug. 11, 2017.

The present invention relates to crystalline forms of carbetocin, amethod of their manufacture, and pharmaceutical compositions thereof.

BACKGROUND

Carbetocin [(also known as1-desamino-1-monocarba-2-(O-methyl)-tyrosine)oxytocin or 1-butanoicacid-2-(O-methyl-L-tyrosine)-1-carbaoxytocin] is a long-acting syntheticoligopeptide analogue of oxytocin, with agonist action. Carbetocinincorporates the following replacements relative to oxytocin: a) theamino-group of cysteine (position 1) by a hydrogen atom; b) of itsdisulphide bond by a thioether bond; and c) the hydroxyl group oftyrosine (position 2) by a methyloxyl group. Carbetocin (PABAL®,DURATOCIN®) is currently approved for the prevention of uterine atonyfollowing delivery of the infant by Caesarean section under epidural orspinal anaesthesia. The dosages used for this medical indication arerelatively small, for instance of the order of 100 micrograms givenonce.

Recently, there has been an increased need for oxytocin receptoragonists, particularly carbetocin. For example, oxytocin receptors haverecently been indicated in the treatment of Prader-Willi Syndrome (seeWO2016/044131). Prader-Willi Syndrome is a genetic disordercharacterised by hyperphagia, food seeking behaviour, rapid weight gain,compulsive behaviour and aggression in young children. As described inWO2016/044131, patients treated with carbetocin exhibit statisticallysignificant improvement over placebo treated patients after 15 days inmeasurements of hyperphagia, obsessive compulsive disorder, food seekingbehaviour and clinical global impression. A relatively large amount ofpeptide has to be produced for this indication, because the dosages usedare significantly higher than those used in the treatment of uterineatony, for instance of the order of tens of milligrams per day, and thetreatment is more long term. It would be desirable to produce relativelylarge amounts of carbetocin of high purity for such indications.

The synthesis of peptides may be carried out using solid phase syntheticprocedures, which are well known in the art. Solution phase synthesis isan alternative method, which may be useful for small quantities ofpeptide. This stage of peptide production is known as the “upstreamprocess”, and results in the formation of a crude peptide product.

Following the synthesis of the crude peptide, it is usually necessary toseparate the peptide of interest from various peptide and non-peptideimpurities. This step is known as the purification step.

Many methods of purifying peptides are known in the art. However,peptide purification methods typically include at least onechromatographic step, for example size exclusion chromatography,hydrophobic interaction chromatography, ion exchange chromatography,free-flow-electrophoresis, affinity chromatography, high performanceliquid chromatography (HPLC) etc. The most commonly employed form ofHPLC is “reversed phase” HPLC (also known as RP-HPLC), in which peptideselute with increasing amounts of an organic solvent, such asacetonitrile, according to their hydrophobicity.

Following the purification step, the peptide typically has to beseparated from volatile solvents. This step is known as the isolationstep. Known methods of separating the peptide from the peptide from thesolvents include ultrafiltration and lyophilisation.

Lyophilisation (also known as freeze-drying) comprises a step of rapidfreezing of the peptide-containing solution, typically by immersing acontainer holding the solution in liquid nitrogen. The container issubsequently placed in a vacuum chamber, which comprises a cooling coil.The volatile solvents sublimate in the vacuum. The sublimation processensures that the purified sample is kept cold.

Lyophilisation is the technique most commonly used in the art to isolatepeptides from solution. This is principally because the technique iswell known, reproducible and easy to carry out. Further, the stabilityof peptides is typically increased at low temperatures.

Methods for purification and isolation of carbetocin and relatedpeptides are known in the art:

CN104592362 describes a step of liquid chromatography purification ofcarbetocin followed by lyophilisation. In most cases the liquidchromatography step is HPLC.

WO2015185584 describes the purification and lyophilisation of oxytocinagonists other than carbetocin.

CN102977192 describes a process of purifying carbetocin by combiningliquid chromatography and ion exchange chromatography. Afterpurification, the product goes through the step of desalination andlyophilisation.

CN104744567 describes a process of purifying carbetocin by ion exchangechromatography followed by lyophilisation.

CN101531705 describes a process of purifying carbetocin using reversephase HPLC followed by transforming the product into an acetate saltusing the ion exchange method. After being transformed into the salt,the product is subsequently lyophilised.

WO2009/122285 discloses a method of purifying oxytocin analoguesinvolving an HPLC step followed by a step of lyophilisation. Rudko A Det al. “Crystalline Salts of Oxytocin: X-ray crystallographic data” J.Crystal Growth, vol. 10, no. 3, 1971, pages 260-262 describes thecharacterisation of crystallised oxytocin salts. Bryn S et al.“Pharmaceutical Solids: A Strategic Approach to RegulatoryConsiderations” Pharmaceutical Research, vol. 12, no. 7, 1995, pages945-954 describes the characterisation of pharmaceutical solids.

It can be seen from the above references that there is a strongprejudice in the art towards using lyophilisation as the isolation stepin the synthesis of carbetocin and other oxytocin receptor agonists.

However, there are several problems associated with lyophilisation, forexample, a large amount of time has to be spent processing the peptide,and the cost of the refrigerant and equipment is very high.

These problems may be acceptable when producing a small amount of apeptide. However, when mass producing the peptide, lyophilisationbecomes a “bottleneck” in the production process. Further, theproportion of the overall cost of production spent on lyophilisationincreases with the mass of peptide produced.

Accordingly, there also exists a need in the art for an improved methodof isolating, carbetocin to remove the lyophilisation “bottleneck” suchthat larger quantities of carbetocin of sufficient purity may beproduced to meet the need in indications such as treatment ofPrader-Willi Syndrome.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to carbetocin incrystalline form.

In a further aspect, the present invention relates to a method ofmanufacturing carbetocin in a crystalline form comprising the step ofcrystallising carbetocin.

In a further aspect, the present invention relates to a pharmaceuticalcomposition comprising carbetocin according to the present invention, orcarbetocin made according the present invention.

FIGURES RELATED TO THE PRESENT INVENTION

The drawings related to the present invention are described below:

FIG. 1 shows the X-ray diffraction pattern (Cu) of solvated crystallineForm I carbetocin;

FIG. 2 shows the X-ray diffraction pattern (Cu) of desolvatedcrystalline Form II carbetocin;

FIG. 3A and FIG. 3B show HPLC chromatograms of solids isolated fromExample 1 (FIG. 3A); and solids isolated from Example 4 (FIG. 3B).

FIG. 4A and FIG. 4B show TG/DTA data relating to solvated crystallineForm I carbetocin from Example 1 (FIG. 4A) and desolvated crystallineform II carbetocin from Example 4 (FIG. 4B).

FIG. 5 shows the X-ray diffraction pattern (Cu-Kα₁) of crystallinecarbetocin obtained in Example 5;

FIG. 6 shows the HPLC Chromatogram of Solids Isolated from Example 5;

FIG. 7 shows differential scanning calorimetry (DSC) data relating tocrystalline carbetocin isolated from Example 5;

FIG. 8A and FIG. 8B show gravimetric water sorption (GVS) data fromcrystalline carbetocin isolated from Example 5: change in mass plot(FIG. 8A) and isotherm plot (FIG. 8B).

DETAILED DESCRIPTION

Carbetocin has not previously been known to form crystals. The presentapplicant surprisingly found that it was possible to form threecrystalline forms of carbetocin, as described herein, two of which maybe denoted Form I and Form II. Form I is solvated (for examplehydrated), while Form II is desolvated. Form II has high stability (seeFIG. 4B) and has an acceptably low ethylene glycol content (see Example3), and may be used, for example, as a medicament. By acceptably lowethylene glycol content, it is meant an ethylene glycol content belowthe ICH limit of 620 ppm, as determined by gas chromatography. Form Imay be used as a synthetic intermediate in the production of Form II. Athird crystalline form is also described herein (see Example 5).

According to the present invention, from a first aspect, there isprovided carbetocin in crystalline form. From a second aspect, there isprovided carbetocin in a solvated (for example hydrated) crystallineform. From a third aspect, there is provided carbetocin in a desolvatedcrystalline form.

By solvated it is meant that the crystalline structure includes eitherordered or disordered solvent molecules. By disordered it is meant thatthe positions of the solvent molecules or the positions of atoms thereinmay vary within the crystal structure. The solvent molecules may beliquid or gaseous at room temperature and atmospheric pressure. Thesolvent molecules may consist of molecules of only one type.Alternatively, the solvent molecules may consist of two or moredifferent types of molecules (one of which may optionally be water).There may be at least 0.1 or more solvent molecules per molecule ofcarbetocin, for example at least 0.2 solvent molecules per molecule ofcarbetocin, for example at least 0.5 solvent molecules per molecule ofcarbetocin, for example at least 1 solvent molecule per molecule ofcarbetocin, for example at least 2 solvent molecules per molecule ofcarbetocin, for example at least 5 solvent molecules per molecule ofcarbetocin. As such, the carbetocin in a solvated crystalline form maybe in the form of a mono-, di-, tri-, tetra-, penta-, or hexa-hydratesolvated crystalline form. Preferably, when the carbetocin is insolvated crystalline form, the solvated crystalline form is amonohydrate or a pentahydrate. Accordingly, in one aspect, carbetocin isin a monohydrate or pentahydrate crystalline form. Such carbetocin mayinclude either ordered or disordered solvent molecules. It is believedthat the number of solvent molecules does not affect whether they areordered or disordered.

By desolvated it is meant that the crystalline structure includes littleor no ordered or disordered solvent molecules. There may be less than orequal to 2 solvent molecules per molecule of carbetocin, for exampleless than or equal to 1 solvent molecule per molecule of carbetocin, forexample less than or equal to 0.5 solvent molecules per molecule ofcarbetocin, for example less than or equal to 0.2 solvent molecules permolecule of carbetocin, for example less than or equal to 0.1 solventmolecules per molecule of carbetocin, for example less than or equal to0.05 solvent molecules per molecule of carbetocin, for example less thanor equal to 0.02 solvent molecules per molecule of carbetocin, forexample less than or equal to 0.01 solvent molecules per molecule ofcarbetocin.

In order to determine the crystalline form, X-ray powder diffraction(XRPD) analysis can be carried out. In the present invention, XRPDanalysis was carried out using Cu K radiation (α₁ λ=1.54060 Å;α₂=1.54443 Å; β=1.39225 Å; α₁:α₂ ratio=0.5) on a PANalytical X'pert pro,as further detailed in Example 1. The carbetocin in crystalline formand/or the carbetocin in a solvated crystalline form may becharacterised by X-ray powder diffraction peaks at about 4.83, 7.43,9.20, 17.87, 19.60, 20.43 and 21.34 degrees 2θ (Cu), and/or becharacterised by an X-ray powder diffraction (Cu) pattern substantiallyas illustrated in FIG. 1 , and/or characterised by having 5 or more, 6or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 ormore, 13 or more, 14 or more, 15 or more, or substantially all of the(Cu) X-ray powder diffraction peaks as shown in Table 1. Accordingly, inone aspect, the carbetocin in crystalline form is characterised by X-raypowder diffraction peaks at about 4.83, 7.43, 9.20, 17.87, 19.60, 20.43,and 21.34 degrees 2θ carried out using Cu K radiation (α₁ λ=1.54060 Å;α₂=1.54443 Å; β=1.39225 Å; α₁:α₂ ratio=0.5).

The carbetocin in crystalline form and/or the carbetocin in andesolvated crystalline form may be characterised by X-ray powderdiffraction peaks at about 4.11, 4.39, 5.60, 7.45, 17.75, 19.16 and19.45 degrees 2θ (Cu) and/or be characterised by an X-ray powderdiffraction (Cu) pattern substantially as illustrated in FIG. 2 , and/orcharacterised by having 5 or more, 6 or more, 7 or more, 8 or more, 9 ormore, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 ormore, or substantially all of the (Cu) X-ray powder diffraction peaks asshown in Table 2. Accordingly, in one aspect, the carbetocin incrystalline form is characterised by X-ray powder diffraction peaks atabout 4.11, 4.39, 5.60, 7.45, 17.75, 19.16 and 19.454 degrees 2θ carriedout using Cu K radiation (α₁ λ=1.54060 Å; α₂=1.54443 Å; β=1.39225 Å;α₁:α₂ ratio=0.5).

The carbetocin in crystalline form may be characterised by X-ray powderdiffraction peaks at about 4.34, 6.43, 8.66, 17.37, 19.03, and 19.39degrees 2θ (Cu-Kα₁) and/or be characterised by an X-ray powderdiffraction (Cu-Kα₁) pattern substantially as illustrated in FIG. 5 ,and/or characterised by having 5 or more, 6 or more, 7 or more, 8 ormore, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 ormore, 15 or more, or substantially all of the (Cu-Kα₁) X-ray powderdiffraction peaks as shown in Table 3. Accordingly, in one aspect, thecarbetocin in crystalline form is characterised by X-ray powderdiffraction peaks at about 4.34, 6.43, 8.66, 17.37, 19.03, and 19.39degrees 2θ carried out using Cu Kα₁ radiation (α₁ λ=1.54060 Å).

TABLE 1 (Cu-K) XRPD peak table for carbetocin in Form I. Boldhighlighted peaks correspond to those being the highest peaks asidentified in Figure 1 (Examples 1 and 2). Area Rel. Pos. FWHM [cts *Backgr. d-spacing Height Int. No. [° 2 Th.] [° 2 Th.] ° 2 Th.] [cts] [Å][cts] [%] 1 3.65 0.15 12.77 532.33 24.19 84.36 2.65 2 4.83 0.06 171.97431.53 18.30 2725.63 85.47 3 6.13 0.06 92.88 372.00 14.41 1472.12 46.164 7.43 0.06 201.21 347.68 11.90 3189.14 100.00 5 9.20 0.06 138.54 378.009.61 2195.83 68.85 6 9.69 0.05 31.51 393.01 9.13 624.18 19.57 7 11.540.15 35.71 427.00 7.67 235.80 7.39 8 12.88 0.08 39.37 421.00 6.87 519.9516.30 9 14.56 0.08 46.76 384.19 6.08 617.61 19.37 10 14.82 0.09 31.97378.00 5.98 361.95 11.35 11 15.91 0.06 41.67 361.00 5.57 660.49 20.71 1217.17 0.08 51.12 380.00 5.16 675.24 21.17 13 17.35 0.08 49.08 384.005.11 648.19 20.33 14 17.87 0.09 182.84 392.21 4.96 2069.98 64.91 1518.52 0.09 78.64 407.00 4.79 890.24 27.91 16 19.32 0.09 60.92 425.724.59 689.67 21.63 17 19.60 0.13 183.83 430.00 4.53 1456.81 45.68 1820.43 0.08 110.35 436.00 4.35 1457.52 45.70 19 21.00 0.13 78.71 435.004.23 623.78 19.56 20 21.34 0.10 181.55 432.00 4.16 1798.48 56.39 2121.68 0.10 70.60 429.00 4.10 699.32 21.93 22 22.18 0.13 34.30 427.004.01 271.82 8.52 23 22.69 0.08 67.05 428.00 3.92 885.61 27.77 24 23.160.13 49.67 426.00 3.84 393.60 12.34 25 23.53 0.08 43.92 422.00 3.78580.07 18.19 26 24.00 0.08 18.57 415.00 3.71 245.25 7.69 27 24.38 0.1024.25 407.00 3.65 240.19 7.53 28 24.77 0.10 22.01 397.82 3.59 218.036.84 29 25.16 0.18 73.75 385.57 3.54 417.45 13.09 30 26.01 0.26 61.97356.00 3.43 245.56 7.70 31 26.33 0.10 15.08 344.00 3.39 149.38 4.68 3227.02 0.15 39.17 314.12 3.30 258.68 8.11 33 28.00 0.10 42.85 289.00 3.19424.44 13.31 34 28.95 0.15 15.10 287.00 3.08 99.73 3.13 35 29.47 0.1012.50 282.00 3.03 123.80 3.88 36 30.14 0.13 27.59 272.00 2.97 218.686.86 37 31.53 0.15 44.27 255.00 2.84 292.33 9.17 38 32.07 0.15 14.80249.00 2.79 97.75 3.07 39 33.16 0.15 15.43 238.00 2.70 101.91 3.20 4034.17 0.20 16.62 260.00 2.62 82.33 2.58 41 34.74 0.18 26.54 275.00 2.58150.26 4.71

TABLE 2 (Cu-K) XRPD peak table for carbetocin in Form II. Boldhighlighted peaks correspond to those being the highest peaks asidentified in Figure 2 (Examples 3 and 4). Pos. FWHM Area Rel. [° 2 [° 2[cts * Backgr. d-spacing Height Int. No. Th.] Th.] ° 2 Th.] [cts] [Å][cts] [%] 1 4.11 0.06 92.55 706.02 21.49 1466.93 64.40 2 4.39 0.06143.71 676.49 20.14 2277.70 100.00 3 5.00 0.10 21.15 608.61 17.67 209.559.20 4 5.60 0.06 59.10 539.20 15.79 936.67 41.12 5 6.58 0.20 21.43439.00 13.43 106.14 4.66 6 7.45 0.09 101.80 400.58 11.87 1152.49 50.60 78.22 0.08 25.45 359.83 10.75 336.08 14.76 8 9.45 0.10 36.87 343.00 9.36365.24 16.04 9 10.67 0.10 15.60 344.00 8.29 154.56 6.79 10 12.99 0.1522.36 397.00 6.82 147.70 6.48 11 13.63 0.13 32.38 401.25 6.50 256.5711.26 12 15.41 0.13 36.79 413.43 5.75 291.56 12.80 13 17.75 0.12 119.81435.00 5.00 1054.98 46.32 14 18.12 0.12 54.63 437.00 4.90 481.03 21.1215 18.48 0.15 54.81 438.00 4.80 361.98 15.89 16 19.16 0.15 141.75 435.004.63 936.10 41.10 17 19.45 0.12 139.15 433.00 4.56 1225.27 53.79 1820.04 0.23 56.25 425.00 4.43 247.66 10.87 19 20.52 0.23 60.38 415.004.33 265.85 11.67 20 20.93 0.18 107.59 405.00 4.24 609.01 26.74 21 22.440.41 57.10 363.05 3.96 141.40 6.21 22 23.49 0.26 48.64 344.00 3.79192.72 8.46 23 24.47 0.18 53.19 342.00 3.64 301.06 13.22 24 28.25 1.2343.87 279.93 3.16 36.21 1.59 25 32.01 0.41 21.63 210.00 2.80 53.56 2.35

TABLE 3 (Cu-Kα1) XRPD peak table for carbetocin in crystalline formobtained as described in Example 5 (Form III). No. Pos. [°2 Th.]d-spacing [Å] Height [cts] Rel. Int. [%] 1  4.3370 20.25752  6599.5214.31  2  6.4326 13.72947  3839.05 8.82 3  8.0491 10.97542   278.34 0.604  8.6648 10.19684  46121.51  100.0   5 10.2212 8.64745  643.40 1.40 610.3427 8.54606 1962.68 4.26 7 11.2271 7.87483  654.49 1.42 8 11.82517.47788 1174.88 2.55 9 12.1233 7.29461  856.51 1.86 10 13.0027 6.80318 394.87 0.86 11 13.2334 6.68508  226.41 0.49 12 13.6140 4.49901 1647.023.57 13 13.8708 6.37926  519.06 1.13 14 14.0103 6.31609 1269.77 2.75 1514.5907 6.06611 1244.01 2.70 16 14.9012 5.94041 2710.87 5.88 17 15.00965.89774 1766.15 3.83 18 14.4094 5.74560 1182.73 2.56 19 16.1421 5.48642 413.17 0.90 20 16.8619 5.25382  858.21 1.86 21 17.3665 5.10226 4458.239.67 22 18.0048 4.92280 2842.92 6.16 23 18.4672 4.80057  720.50 1.56 2418.7031 4.74055  404.75 0.88 25 19.0319 4.65939 3572.38 7.75 26 19.39184.57371 3844.17 8.33 27 19.7778 4.48530 1577.37 3.42 28 20.2075 4.39089 774.88 1.68 29 20.5431 4.31990  925.40 2.01 30 20.7560 4.27609 1394.513.02 31 21.0184 4.22328 2728.11 5.92 32 21.7698 4.07918 2143.36 4.65 3322.1539 4.00932 1416.60 3.07 34 22.5554 3.93886  605.37 1.31 35 23.22903.82613 2097.57 4.55 36 23.7449 3.74416  396.84 0.86 37 24.3125 3.65802 690.11 1.50 38 24.5465 3.62368  629.24 1.36 39 24.8555 3.57932  578.441.25 40 25.0834 3.54732 1119.99 2.43 41 25.7775 3.45335  406.85 0.88 4226.1605 3.40365  804.34 1.74 43 26.6344 3.34416  270.06 0.59 44 27.10683.28694  596.21 1.29 45 28.0519 3.17831  434.48 0.94 46 28.2113 3.16072 543.61 1.18 47 29.1046 3.06570  533.43 1.16 48 29.4103 3.03452  272.680.59 49 29.7218 3.00343  765.65 1.66 50 30.0597 2.97044  271.38 0.59 Thebold highlighted peaks correspond to those being the highest peaksidentified in FIG. 5, Example 5.

Table 3 shows the data obtained from Example 5. It will be noted thatthe radiation source for the values as reported in Table 3 is a Cu-Kα₁source, whereas the radiation source for the values as reported inTables 1 and 2 is a Cu—K source. The above XPRD peak table, Table 3(FIG. 5 , Example 5), nevertheless indicates carbetocin of a differentcrystal form or polymorph when compared to Examples 1 and 2 (FIG. 1 ,Table 1) and Examples 3 and 4 (FIG. 2 , Table 2).

According to the present invention in a further aspect, there isprovided a method of manufacturing carbetocin in a crystalline form, themethod comprising a step of crystallising carbetocin.

By crystallising it is meant the process of forming a crystalline formof carbetocin from carbetocin dissolved in a solvent. By crystallineform is meant a solid material with a regularly repeating internalarrangement of atoms and external plane faces. Crystalline forms may bedistinguished from amorphous forms on the basis of X-ray powderdiffraction analysis. Crystalline forms are characterised by X-raypowder diffraction peaks as described herein. In amorphous solid formsthe XPRD pattern is essentially continuous in appearance, i.e. withoutdistinct peaks.

The carbetocin in a crystalline form may be crystallised from a mixturecomprising carbetocin and one or more liquid(s), the one or moreliquid(s) optionally comprising one or more liquid(s) from the groupconsisting of water, aqueous acetate buffer, ethylene glycol,acetonitrile, ethanol, methanol, propanol, isopropanol, 1,2-propanedioland dimethylformamide, for example a mixture of ethylene glycol andacetonitrile, for example a mixture of ethanol, ethylene glycol andacetonitrile, for example a mixture of propanol, ethylene glycol andacetone, for example a mixture of isopropanol, ethylene glycol andacetone, for example a mixture of dimethylformamide, ethylene glycol andacetonitrile, for example a mixture of dimethylformamide andacetonitrile, for example a mixture of dimethylformamide and acetone,for example a mixture of ethanol and acetonitrile, for example a mixtureof methanol and acetonitrile, for example a mixture of 1,2-propanedioland acetonitrile, for example a mixture of 1,2-propanediol and acetone.Where the one or more liquids comprises two or more liquids, one of theliquids of the two or more liquids may be an antisolvent (as definedbelow). The one or more liquid(s) may comprise ethylene glycol andantisolvent in a ratio of from 15:85 to 25:75, for example a ratio offrom 17.5:82.5 to 22.5:77.5, for example a ratio of about 20:80, andwherein the addition of antisolvent alters the ratio of ethylene glycolto acetonitrile to a ratio of from 1:99 to 30:70, for example a ratio offrom 2:98 to 25:75, for example a ratio of from 3:97 to 20:80, forexample a ratio of from 5:95 to 20:80, for example a ratio of from 5:95to 15:85, for example a ratio of from 7.5:92.5 to 12.5:87.5, for examplea ratio of about 10:90, for example a ratio of from 5:95 to 10:90, forexample a ratio of 5:95 to 7.5:92.5, for example a ratio of about6.7:93.3.

The one or more liquid(s) may comprise a mixture of ethylene glycol andacetonitrile.

The one or more liquid(s) may comprise ethylene glycol and acetonitrilein a ratio of from 1:99 to 50:50, for example a ratio of from 2:98 to40:60, for example a ratio of from 3:97 to 35:65, for example a ratio offrom 5:95 to 35:65, for example a ratio of from 8:92 to 30:70, forexample a ratio of from 10:90 to 30:70, for example a ratio of from15:85 to 25:75, for example a ratio of from 17.5:82.5 to 22.5:77.5, forexample a ratio of about 20:80.

The one or more liquid(s) may comprise water. The one or more liquid(s)may comprise aqueous acetate buffer. In a particular aspect, thecarbetocin in crystalline form is crystallised from a mixture comprisingcarbetocin and one or more liquid(s), the one or more liquid(s)comprising one or both of water and aqueous acetate buffer.

In one embodiment, the one or more liquid(s) may be water. If the one ormore liquid(s) is water, the water may have a pH of between about 2 and6, preferably a pH between about 3 and 4, more preferably a pH of about3.5.

In one embodiment, the one or more liquid(s) may be aqueous acetatebuffer. The aqueous acetate buffer may be formed from an aqueous mixtureof acetic acid and an acetate salt. The acetate salt may be any salt ofacetate with a suitable counterion. A suitable counterion may be, forinstance, an alkali metal ion, an alkaline earth metal ion or an organiccation. The counterion may be lithium, sodium, potassium, magnesium,calcium or ammonium. Preferably, the counterion may be sodium orpotassium, most preferably, the counterion may be sodium. Preferably,the aqueous acetate buffer has a pH of between about 4 and 7, preferablya pH between about 5 and 6, most preferably the pH is about 5.5.Preferably, the aqueous acetate buffer has a concentration of between 20and 30 mM, most preferably the concentration is about 25 mM.

The carbetocin in crystalline form may be obtained by cooling themixture comprising carbetocin and the one or more liquid(s), for examplefrom 40° C. to 5° C., or by cycling the temperature of the mixturecomprising carbetocin and one or more liquid(s), for example between 40°C. and 5° C. The temperature may be changed at a rate of 5° C. to 50° C.per hour, such as 35° C. per hour. By cycling the temperature of themixture it is meant that the temperature must be subsequently loweredthen raised, or vice versa. The temperature may be lowered then raised,or vice versa, two or more times, for example three or more times, forexample four or more times, for example five or more times, for exampleten or more times. Following cooling or cycling of the temperature ofthe mixture comprising carbetocin and one or more liquids, thetemperature may be maintained, such as at 5° C., for a suitable lengthof time to form crystalline carbetocin. Typically, crystallinecarbetocin may form and may be isolated within 6 hours to 24 hours, suchas at about 12 hours or at about 18 hours. The cooling, cycling ormaintenance of the temperature may occur with or without agitation ofthe mixture.

Alternatively, the carbetocin in crystalline form may unexpectedly beobtained by maintaining a mixture comprising carbetocin and water, or amixture comprising carbetocin and aqueous acetate buffer, at atemperature of at least 15° C., such as 20° C., such as 30° C., such as40° C. for a suitable length of time to form crystalline carbetocin.Typically, crystalline carbetocin forms between 3 to 100 days, moretypically between 3 to 60 days, most typically between 7 to 12 days. Inone alternative embodiment, the carbetocin in crystalline form may beobtained by maintaining a mixture comprising carbetocin and water, or amixture comprising carbetocin and aqueous acetate buffer, at atemperature of 20° C., for about 3 to 60 days. In another alternativeembodiment, the carbetocin in crystalline form may be obtained bymaintaining a mixture comprising carbetocin and water, or a mixturecomprising carbetocin and aqueous acetate buffer, at a temperature of40° C. for about 7 to 12 days. In these alternative embodiments, allother steps described below, with the exception of the step related toaddition of an antisolvent, may be performed.

The carbetocin may be crystallised from a mixture containing at leastone solvent and at least one antisolvent. By solvent is meant a liquidin which carbetocin is readily dissolves or is readily soluble. Thesolvent may be any solvent in which carbetocin is soluble in an amountat is soluble in an amount at standard conditions of 0.01 mg/ml orgreater, for example 0.05 mg/ml or greater, for example 0.1 mg/ml orgreater, for example 0.5 mg/ml or greater, for example 1 mg/ml orgreater, for example 5 mg/ml or greater, for example 10 mg/ml orgreater, for example 20 mg/ml or greater. By antisolvent is meant aliquid in which carbetocin dissolves less readily relative to a solvent,or in which carbetocin is, relatively to a solvent, less soluble. Theantisolvent may be selected relatively to the solvent and may be anysolvent in which carbetocin is soluble in an amount at standardconditions of less than 20 mg/ml, for example less than 10 mg/ml, forexample less than 5 mg/ml, for example less than 1 mg/ml, for exampleless than 0.5 mg/ml, for example less than 0.1 mg/ml, for example lessthan 0.05 mg/ml, for example less than 0.01 mg/ml. It will be understoodby the skilled person that when carbetocin is soluble in an amount of,for example, 10 mg/ml or greater in a solvent, in an antisolvent it willbe less soluble, i.e. it will be soluble in an amount of less than 10mg/ml, for example less than 5 mg/ml, for example less than 1 mg/ml, forexample less than 0.5 mg/ml, for example less than 0.1 mg/ml, forexample less than 0.05 mg/ml, for example less than 0.01 mg/ml. Unlessotherwise specified, the terms solvent and antisolvent refer to thesolubility behaviour of carbetocin at room temperature and atmosphericpressure.

The solvent may comprise one or more liquid(s) from the group consistingof water, aqueous acetate buffer, ethylene glycol, ethanol, methanol,propanol, isopropanol, and 1,2-propanediol, The solvent may have arelative polarity index (RPI), as described by Christian Reichardt(Solvents and Solvent Effects in Organic Chemistry, Wiley-VCHPublishers, 3^(rd) ed., 2003) of greater than 0.5, for example greaterthan 0.6, for example greater than 0.7, for example greater than 0.8,for example greater than 0.9, for example greater than 1.0. The solventmay be or comprise any one or more of water, an aqueous acetate buffersolution, or an alcohol, for example any one or more of, water(RPI=1.000), an aqueous acetate buffer solution, ethylene glycol(RPI=0.790). ethanol (RPI=0.654), methanol (RPI=0.762), propanol(RPI=0.803), isopropanol (RPI=0.787), or 1,2-propanediol (RPI=0.72).

The carbetocin in a crystalline form may be crystallised from a mixturecomprising carbetocin and one or more liquid(s), the carbetocin beingpresent in a solvent at a concentration of from about 1 mg/ml to 200mg/ml, preferably from about 10 mg/ml to 150 mg/ml, most preferably fromabout 20 mg/ml to 100 mg/ml. In one embodiment, the solvent may bewater. In one embodiment, the solvent may be aqueous acetate buffer. Inone embodiment, the solvent may be ethylene glycol.

The method may comprise a further step of adding an antisolvent to themixture comprising carbetocin and one or more liquid(s), for exampleadding the antisolvent prior to cooling the mixture.

The antisolvent may have a relative polarity index (RPI), as describedby Christian Reichardt (Solvents and Solvent Effects in OrganicChemistry, Wiley-VCH Publishers, 3^(rd) ed., 2003) of less than 1, forexample less than 0.9, for example less than 0.8, for example less than0.75, for example less than 0.7, for example less than 0.6, for exampleless than 0.5. The antisolvent may be or comprise any one or more of, anester, a ketone, a nitrile, or an ether, for example any one or more of,acetonitrile (RPI=0.460), ethyl acetate (RPI=0.228), acetone(RPI=0.355), or methyl tert-butyl ether (RPI=0.124).

Thus, in one embodiment of the method, carbetocin may be crystallisedfrom a mixture comprising carbetocin and one or more liquid(s), the oneor more liquid(s) comprising ethylene glycol and acetonitrile. Thecarbetocin may be present in the mixture comprising carbetocin and oneor more liquid(s) at a concentration of from 10 mg/ml to 150 mg/ml, mostpreferably at about 100 mg/ml. The ethylene glycol and acetonitrile maybe present in a ratio of from 5:95 to 35:65. The carbetocin incrystalline form may be obtained by cooling the mixture comprisingcarbetocin and the one or more liquid(s) from 40° C. to 5° C. at a rateof 35° C. per hour and maintaining the temperature at 5° C. for asuitable length of time to form crystalline carbetocin, such as forabout 12 hours or for about 18 hours.

The method may comprise a further step of seeding the mixture comprisingcarbetocin and one or more liquid(s) with a crystal, for example acarbetocin crystal, for example a crystal of the solvated crystallineForm I of carbetocin.

By seeding is meant adding homogeneous or heterogeneous crystals, i.e.seed crystals, to the mixture to nucleate and/or grow additionalcarbetocin in a crystalline form. By homogeneous crystals is meantcrystalline carbetocin in either of its forms. By heterogeneous crystalsis meant crystals of another material.

The method may comprise a further step of inducing crystallisation inthe mixture comprising carbetocin and one or more liquid(s).Crystallisation may be induced by any suitable means to promotenucleation and growth of crystals, for example by disturbing the surfaceof the mixture comprising carbetocin and one or more liquid(s) to createseed crystals, such as by pipetting liquid up and down from the surfaceof the mixture comprising carbetocin and one or more liquid(s) orscratching where the surface of the mixture comprising carbetocin andone or more liquid(s) meets the surface of the container in which themixture is held.

The method may comprise a further step of desolvating and optionallydrying the carbetocin in a crystalline form.

By desolvating is meant removal of some or substantially all of thesolvated molecules from the crystalline structure of carbetocin, suchthat the crystalline structure includes little or no ordered ordisordered solvent molecules. In a preferred embodiment, desolvatingmeans converting carbetocin from a pentahydrate crystalline form to amonohydrate crystalline form.

The desolvation of the carbetocin in a crystalline form may be carriedout by washing the carbetocin in a crystalline form in an antisolvent,for example acetonitrile, optionally at a temperature at or below 20°C., for example from −30° C. to 20° C., for example from −20° C. to 20°C., for example from −10° C. to 20° C., for example from −5° C. to 15°C., for example from 0° C. to 10° C., for example about 5° C., and thendrying, for example drying under vacuum. The drying may occur undervacuum for a suitable length of time to effect the desolvation, such asfor greater than 1 hour, such as for about 24 hours. Preferably, thecarbetocin in a crystalline form may be washed in acetonitrile at atemperature of about 5° C. and dried under vacuum at a temperature ofabout 20° C. for about 24 hours to effect desolvation.

Desolvation may also be carried out by heating the carbetocin in acrystalline form to a temperature of at least 40° C. to at most 190° C.,or by exposing carbetocin in a crystalline form to an environment of lowrelative humidity, such as 40% relative humidity or less.

Thus, in one embodiment of the method, carbetocin may be crystallisedfrom a mixture comprising carbetocin and one or more liquid(s), the oneor more liquid(s) comprising ethylene glycol and acetonitrile. Thecarbetocin may be present in the mixture comprising carbetocin and oneor more liquid(s) at a concentration of from 10 mg/ml to 150 mg/ml, mostpreferably at about 100 mg/ml. The ethylene glycol and acetonitrile maybe present in a ratio of from 5:95 to 35:65. Additional antisolvent,such as acetonitrile, may be added. The carbetocin in crystalline formmay be obtained by cooling the mixture comprising carbetocin and the oneor more liquid(s) from 40° C. to 5° C. at a rate of 35° C. per hour andmaintaining the temperature at 5° C. for a suitable length of time toisolate crystalline carbetocin, such as for about 12 hours or for about18 hours. The carbetocin in a crystalline form may be washed inacetonitrile at a temperature of about 5° C. and dried under vacuum at atemperature of about 20° C. for about 24 hours to effect thedesolvation.

Optionally, a filtration step can be carried out prior tocrystallisation. The filtration step preferably comprises filtration bycentrifugation. Accordingly, in one aspect, the method of manufacturingcarbetocin in a crystalline form comprises the steps of (1) filtration,preferably by centrifugation; and (2) crystallisation.

Optionally, a washing step can be carried out prior to crystallisation.For example, the carbetocin, for example crude carbetocin, may beslurried, for example slurried in acetonitrile, for example slurried inacetonitrile for 2 hours to a week, for example slurried in acetonitrilefor about 18 hours with constant agitation. Washing crude carbetocinincreases the purity prior to crystallisation by approximately 1 to 2%and significantly increases the assay values from around 44% to around70% (in acetonitrile). Accordingly, in one aspect, the method ofmanufacturing carbetocin in a crystalline from comprises the steps of(1) washing carbetocin, for example crude carbetocin, in acetonitrile;and (2) crystallisation. In another aspect, the method of manufacturingcarbetocin in crystalline form comprises the steps of (1) washingcarbetocin, for example crude carbetocin, in acetonitrile; (2)filtration, preferably by centrifugation; and (3) crystallisation.

The applicants have advantageously and surprisingly found that it ispossible to isolate carbetocin without any requirement forlyophilisation by crystallising the carbetocin, for examplecrystallising the carbetocin from solution.

The method provides high purity carbetocin in an acceptable yieldwithout the need for a lyophilisation step.

The carbetocin in the mixture comprising carbetocin and one or moreliquid may be substantially pure carbetocin, or alternatively may becrude carbetocin.

Herein, the term “crude” as in, “crude carbetocin” means carbetocinwhich is insufficiently pure to be used as a pharmaceutical product. Thecrude peptide may have a purity of less than 95%, for example less than92.5%, for example from 90% to 93%, for example from 91% to 93%, asmeasured by UV-HPLC. The impurities found in the crude peptide mayinclude one or more of inorganics, residual solvent (for example DMF),peptide-related impurities and residual peptide coupling reagents.

The (product) carbetocin in crystalline form/carbetocin in solvated (forexample hydrated) crystalline form/carbetocin in desolvated crystallineform may have purity greater than or equal to 95%.

The crude carbetocin may be synthesised by methods well known in theart, for example the methods analogous to those described inWO2009/122285 (International Patent Application No. PCT/IB2009/005351)of Ferring B.V.

According to the invention in a further aspect there is provided apharmaceutical composition including carbetocin according to theinvention, or carbetocin made according to a method of the invention.The pharmaceutical composition of the invention may be for use as amedicament. The pharmaceutical composition of the invention may be foruse in the treatment of a neurological disorder or reproductivedisorder, for example for use in the treatment of Prader-Willi syndrome(as described in WO2016/044131 (International Patent Application No.PCT/US2015/04911) of Ferring B.V.); or for example for use in thetreatment or prevention of uterine atony, for example following vaginaldelivery of the infant, delivery of the infant by Caesarean section; orfor use in the treatment or prevention of uterine atony in a patient whois at risk of developing postpartum hemorrhage (PPH); and/or for use inthe treatment or prevention of excessive bleeding following vaginaldelivery (as described in WO2009/122285 (International PatentApplication No. PCT/IB2009/005351) of Ferring B.V).

The present invention is exemplified below. The examples may describepreferred embodiments of the invention but are not meant to be limitingin any way.

Example 1—Preparation of Solvated Crystalline Form I Carbetocin

Step i: Synthesis

Crude carbetocin of purity approx. 91% was obtained by synthetic methodsanalogous to those described in WO2009/122285 (International PatentApplication No. PCT/IB2009/005351) of Ferring B.V.

Step ii: Preparation of the Solution 60 mg crude carbetocin obtained instep i) was dissolved in 0.6 mL of a 30:70 (v/v) mixture of ethyleneglycol (first liquid):acetonitrile (second liquid) at 40° C. The vesselwas then seeded with Form I (solvated) carbetocin crystals. It will beappreciated that seeding is not required, but may expedite thecrystallisation.Step iii: Crystallisation

The solution obtained in step ii) was heated to 40° C. and kept at thistemperature for 30 minutes. The mixture was then filtered bycentrifugation to remove any insoluble impurities. The mixture was thenstirred at 40° C. for thirty minutes, cooled to 5° C. over the course ofone hour, and then held at 5° C. for overnight with constant agitation.

The precipitated material was isolated.

XRPD analysis was carried out on a PANalytical X'pert pro. The sampleswere scanned between 3 and 35° 2θ. The material was gently ground torelease any agglomerates and loaded onto a multi-well plate with Kaptonor Mylar polymer film to support the sample. The multi-well plate wasthen placed into the diffractometer and analysed using Cu K radiation(α₁ λ=1.54060 Å; α₂=1.54443 Å; β=1.39225 Å; α1:α₂ ratio=0.5) running intransmission mode (step size 0.0130° 2θ) using 40 kV/40 mA generatorsettings.

Carbetocin having a X-ray diffraction pattern substantially as shown inTable 1 and FIG. 1 (Form I) crystallised from the solution.

The solids were analysed by TG/DTA for ease of mass loss/thermal events(FIG. 4 a ).

The purity of the carbetocin in the (solvated) crystalline form wascalculated at 96.2% by UV-HPLC (FIG. 3 a ) according to the methodoutlined in Table 4.

TABLE 4 Analytical HPLC parameters for Example 1 Parameter Setting LCHPLC including: Gradient pump UV detector Autosampler Column heater (60°C.) Column Waters XBridge C18, 150 × 2.1 mm, 3.5 μm Mobile phase Aammonium acetate 0.30 g/L in water Mobile phase B 50% Mobile Phase A:50% Acetonitrile (% v/v) Flow rate 0.8 mL/min Injection volume 40 μLDetection UV 220 nm Column 60° C. temperature Autosampler  5° C.temperature Time (min.) % A % B Gradient 0 100 0 20 75 25 21 100 0 25100 0 Sample Crystals dissolved in milli-Q water and diluted preparationto approx. 0.5 mg/mLwith milli-Q. water Evaluation of Purity determinedas relative UV area % of results total area.

Example 2—Preparation of Solvated Crystalline Form I Carbetocin

Step i: Synthesis

Crude carbetocin of purity approx. 91% was obtained by synthetic methodsanalogous to those described in WO2009/122285 (International PatentApplication No. PCT/IB2009/005351) of Ferring B.V.

Step ii: Preparation of the Solution

60 mg crude carbetocin obtained in step i) was dissolved in 0.6 mL of a30:70 (v/v) mixture of ethylene glycol (first liquid):acetonitrile(second liquid) at 40° C. The vessel was then seeded with Form 1(solvated) carbetocin crystals. It will be appreciated that seeding isnot required, but may expedite the crystallisation.

Step iii: Addition of Antisolvent

Sufficient Acetonitrile was added to adjust the ratio of ethyleneglycol:acetonitrile to 6.7:93.3 (v/v)

Step iv: Crystallisation

The solution obtained in step iii) was heated to 40° C. and kept at thistemperature for 30 minutes. The mixture was then filtered bycentrifugation to remove any insoluble impurities. The mixture was thenstirred at 40° C. for thirty minutes, cooled to 5° C. over the course ofone hour, and then held at 5° C. overnight with constant agitation.

The precipitated material was isolated.

XRPD analysis was carried out as described above with respect to Example1.

Carbetocin having a X-ray diffraction pattern substantially as shown inTable 1 and FIG. 1 crystallised from the solution.

Example 3—Preparation of Desolvated Crystalline Form II Carbetocin

In order to remove ethylene glycol present in the crystallised materialproduced by Examples 1 and 2, the crystallised material of Example 1 orExample 2 was desolvated and dried.

Washing the crystallised Form I material in acetonitrile at 5° C.followed by drying under vacuum resulted in the desolvation of solvatedForm I, to produce desolvated Form II crystals, which have a diffractionpattern substantially as shown in Table 2 and FIG. 2 . The Form IIcrystals were found to have ethylene glycol levels below the ICH limitof 620 ppm as determined by gas chromatography, with parameters as inTable 5 below.

TABLE 5 Gas chromatography parameters Column: Agilent J&W D-B-624, 30 m× 0.32 mm, 1.8 μm d.f. Oven Temperature: Initial: 35° C. (hold 1 min)Ramp: 6° C./min to 60° C. (hold 0 min) Ramp: 12° C./min to 225° C. (hold0 min) Injector Temperature 230° C. Injection Pressure 3.7 psi InjectionMode Split Injection Split ratio; Split 10:1; 15.0 mL/min; 22.7 mL/minflow; Total Flow Injection volume 1 μL Detector Temperature 250° C.Detector Hydrogen 30.0 mL/min Detector Air  330 mL/min Make-up Flow 10.0mL/min Make-up Gas Air

Example 4—Preparation of Desolvated Crystalline Form II Carbetocin

To approximately 300 mg of crude carbetocin (purity approximately91.3%), 3 ml of a pre-prepared 30% ethylene glycol:70% acetonitrile(v/v) solvent mixture was added and the mixture heated to 40° C. for 30minutes with constant agitation.

After 30 minutes, the mixture was filtered by centrifugation to removeany insoluble impurities. To this mixture (still at 40° C.), 1.5 mlacetonitrile was added in 0.5 ml aliquots. No precipitation was observedat 40° C., even after the complete addition of the acetonitrile.

The mixture was then stirred at 40° C. for 1 hour, and cooled to 5° C.over the course of 1 hour and then held at 5° C. for 18 hours withconstant agitation.

After 18 hours, precipitated material was isolated, washed withapproximately 5 ml of acetonitrile and then dried under vacuum atambient temperature for 24 hours.

The next day the solids were analysed by HPLC for purity and assay (FIG.3 b ), TG/DTA for ease of mass loss/thermal events (FIG. 4 b ) as wellas polarised light microscopy (PLM) and XRPD for morphology andcrystalline content.

The PLM analysis indicated that the final isolated solid comprised amixture of agglomerates (50-100 μm), that easily dispersed to very smallneedle-like crystals (length <10 μm).

The TG/DTA data for crystals formed by the Example 4 method and shown inFIG. 4 b shows that there was a total mass loss of only approximately0.8% up to 110° C. This was found to be a two-step process, with a firstmass loss of approximately 0.5% up to approximately 60° C., and a secondmass loss of approximately 0.3% up to approximately 110° C. These masslosses correspond to weakly surface bound acetonitrile and water, andare not indicative of any solvation of the crystal form itself.Accordingly, the loss of this solvent does not alter the crystallinityof the desolvated crystalline Form II.

These results indicate that the desolvated crystalline Form IIcarbetocin is highly stable.

Example 5—Preparation of Solvated Crystalline Form I Carbetocin

Step i: Synthesis

Crude carbetocin of purity approx. 93.5% was obtained by syntheticmethods analogous to those described in WO2009/122285 (InternationalPatent Application No. PCT/IB2009/005351) of Ferring B.V.

Step ii: Preparation of the Solution

An acetate buffer, 25 mM, pH 5.5 was prepared from sodium acetatetrihydrate, glacial acetic acid and ultrapure water. 354 mg crudecarbetocin obtained in step i) was dissolved in 16.6 mL of the acetatebuffer. The solution was filtered with a 0.22 μM PVDF syringe filter and500 μL portions of the solution was aliquoted into vials which werethereafter sealed. The pH of the carbetocin solution was 5.3.

Step iii: Crystallisation

The solution obtained in step ii) was heated to 40° C. and kept insealed vials at this temperature. After 3 days the vials were taken outand an Eppendorf glass pipette was used to gently suck the solution upand back in the vials to thereby create some seeds for thecrystallisation. After the pipetting the vials were sealed again andmaintained at 40° C. After 9 days particles with a crystal-likeappearance had formed. The precipitated material was isolated.

XRPD analysis was carried out on a PANalytical X'pert pro with a Cu-Kα₁monochromator (α₁ λ=1.54060 Å). The samples were scanned between 2 and35° 2θ. The material was gently crushed and smeared on a zero-backgroundwafer of Si, which was then placed into a slow spinning sample holder inthe diffractometer running in transmission mode (scan speed 0.01°/s,step size 0.017° 2θ) using 45 kV/40 mA generator settings. Themeasurements were performed using a programmable incident divergencyslit.

The X-ray diffraction pattern of the carbetocin crystals obtained ispresented in Table 3 and FIG. 5 . The X-ray diffraction patternindicates carbetocin with a different crystal form or polymorph whencompared to Examples 1 and 2 (FIG. 1 , Table 1) and Example 3 and 4(FIG. 2 , Table 2).

Differential scanning calorimetry (DSC) analysis was performed on aNetzsch DSC 204F1. Several milligrams of crystals were isolated from themother solution and left to air dry in a fume hood for a few hours atabout 20% relative humidity (RH). The crystals were gently crushed intoa powder material and 1.2 mg of this material was charged into a 25 μLAl pan. A lid was adapted and crimped onto the pan, prior to beingpierced with a pinhole (diameter 0.25 mm). The sample was analysed from20 to 250° C. using a heating rate of 5 K/min.

The DSC data for crystals formed by the Example 5 method and shown inFIG. 7 shows that there is a loss of volatile material in the region of40 to 120° C., corresponding to a loss of weakly surface bound water andsolvated water. A melting endotherm with onset of 192° C. corresponds tothe melting of anhydrous carbetocin.

Gravimetric vapour sorption (GVS) was performed on an SMS DVS-1. 1.4 mgof crystals and powder were added into an Al pan and exposed to stepwiserelative humidity (RH) changes during two consecutivecycles:20-30-40-50-60-70-80-70-60-50-40-30-20-10-0-10-20-30-40-50-60-70-80-90-80-70-60-50-40-30-20-10-0%RH in an open loop mode. The temperature was maintained at 25° C. andpure nitrogen flow rate of 200 ml/min was used. The dm/dt criteriaapplied was 0.001 weight-%/min during a 5 minute window, with a maximumtime of 150 minutes for all steps, with the exception of steps at 0% RHthat were set to 6 hours.

The GVS data are shown in FIG. 8 . A GVS isotherm plot is shown in FIG.8 b . A plateau at around 2% (w/w) corresponds to a monohydrate plussome loosely bound surface water. A second plateau at an additionalapproximately 8% (w/w) corresponds to a pentahydrate with some looselybound surface water. The pentahydrate exists at about 60% RH and above(sorption) and from about 40 to 90% RH (desorption).

The purity of the carbetocin in the (solvated) crystalline form wascalculated at 98.7% by UV-HPLC according to the method outlined in Table6.

TABLE 6 Analytical HPLC parameters for Example 5 Parameter Setting LCHPLC including: Gradient pump UV detector Autosampler Column heater (60°C.) Column Waters XBridge C18, 150 × 2.1 mm, 3.5 μm Mobile phase Aammonium acetate 0.15 g/L in 19% MeCN (acetonitrile) in water Mobilephase B ammonium acetate 0.075 g/L in ~60% MeCN (acetonitrile) in waterFlow rate 0.4 mL/min. Injection volume 20 μL Detection UV 220 nm Columntemperature 60° C. Autosampler  5° C. temperature Time (min.) % A % BGradient 0 100 0 20 75 25 30 0 100 31 100 0 40 100 0 Sample preparationCrystals dissolved in milli-Q water and diluted to approx. 0.1 mg/mLwithmilli-Q. water Evaluation of results Purity determined as relative UVarea % of total area.

What is claimed is:
 1. Carbetocin in a solvated crystalline form,wherein the carbetocin is in solvated crystalline Form III.
 2. Apharmaceutical composition comprising the carbetocin in solvatedcrystalline form according to claim
 1. 3. A method of treatment of aneurological disorder or reproductive disorder comprising administeringthe pharmaceutical composition of claim 2 to a subject in need thereof.4. The method of claim 3, wherein the neurological disorder orreproductive disorder is selected from one or more of male or femalesexual dysfunction, Prader-Willi syndrome, uterine atony, and excessivebleeding following vaginal delivery.
 5. A method of manufacturing thecarbetocin in solvated crystalline form of claim 1, the methodcomprising a step of crystallizing carbetocin.
 6. A method according toclaim 5, wherein the carbetocin is crystallized from a mixturecomprising carbetocin and one or more liquid(s), wherein the one or moreliquid(s) comprise one or both of water and aqueous acetate buffer.
 7. Amethod according to claim 5, wherein the carbetocin is crystallized froma mixture comprising carbetocin and one or more liquid(s), wherein theone or more liquid(s) comprise one or more selected from the groupconsisting of ethylene glycol, acetonitrile, ethanol, methanol,propanol, isopropanol, 1,2-propanediol and dimethylformamide.
 8. Amethod according to claim 7, wherein the one or more liquid(s) compriseethylene glycol, acetonitrile, and ethanol.
 9. A method according toclaim 7, comprising cooling or cycling the temperature of the mixturecomprising the carbetocin and one or more liquid(s), optionally from 40°C. to 5° C.
 10. A method according to claim 7, further comprising addingan antisolvent to the mixture comprising the carbetocin and one or moreliquid(s).
 11. A method according to claim 10, wherein the antisolventis acetonitrile.
 12. A method according to claim 7, further comprisingmaintaining the mixture comprising the carbetocin and one or moreliquid(s) at a temperature of at least 15° C. for 3 to 100 days.
 13. Amethod according to claim 7, further comprising seeding the mixturecomprising the carbetocin and one or more liquid(s) with a crystal.